Metastasis inducing DNA&#39;s

ABSTRACT

The invention relates to metastasis inducing DNA&#39;s, a method of identifying such DNA&#39;s and their use in diagnosis and therapy. It includes a method of screening and recovering Met-DNA comprising the steps of: (1) transferring fragments of human DNA from malignant, metastatic cancer cells into a cell line that produces only benign, non-metastasizing tumours when injected into a syngeneic animal; (2) injecting the transformed cells into a syngeneic animal; (3) selecting those animals in which metastasizing tumours have been identified; and (4) recovering the Met-DNA therefrom.

The present invention relates to metastasis inducing DNA's, a method ofidentifying such DNA's, and their use in diagnosis and therapy.

Most cancers are thought to be due to alterations in specific genescaused either by mutation making their gene-product in some way moreeffective or by over expression of a normal gene giving an enhancedeffect. These oncogenes have largely been identified by introducinggene-length fragments of DNA from human cancers into a mouse fibroblastcell line, in culture, and selecting those cell lines that grow in anuncontrolled manner in liquid or semi-solid medium. The oncogenesthemselves have been isolated by cloning the human DNA fragments awayfrom the mouse DNA by standard recombination techniques. Alternativelymutations can arise in genes that suppress their own activity such as,for example, p53 or Rb or which suppress the levels of their productssuch as, for example NM-23. These are referred to as tumour suppressoroncogenes. In the commonly-occurring cancers, it is believed thatbetween 5 and 7 such changes in oncogenes or tumour suppressor oncogenesare required to produce a full-blown cancer.

WO 86/03226 discloses a method for detecting a discrete, transmissiblemammalian gene associated with tumour metastasis. The method uses anon-syngeneic system. The teaching was later retracted—Proc Nat. Acad.Sci USA., 1988, 85 5581.

WO 94/28129 identifies a tumour metastasis gene of 2858 base pairs whichcodes for a protein which is expressed in malignant human tumours andtheir metastasis. The method used to identify it used a non-syngeneicsystem employing nude (defective) mice.

Cancer research 54, 2785-2793 (1994) is a paper by the applicants. Itdiscloses a method for showing the presence of metastasis inducing DNA.No disclosure is, however, made of how to recover the sequences foridentification.

Cancer research 54 832-837 (1994) is a paper suggesting that antisenseOPN DNA expression was associated with reduced tumorigenicity of thesecells in the flanks and in lungs. The paper does not measure orinvestigate metastasis as such.

EP 0607054 disclosures a process for constructing a cDNA library. Itdescribed a method, using linkers and PCR for identifying signalpeptides. The application is not to metastasis at all and the approachuses expression vectors for detection.

The major forms of cancer, including breast cancer, lung cancer andcolonic cancer cannot be cured effectively because, although the currenttherapies may be effective against the primary tumours, they are largelyineffective against the disseminating or metastasizing cells, whichultimately kill the patient. Despite the enormous effort in cancerresearch very little is known at the molecular level about the mostimportant life-threatening process, that of metastasis. Most of theoncogenes and suppressor oncogenes that have been discovered have beenfound from their ability to promote uncontrolled growth of the mousefibroblast cell line. The major problem in this field is thatdetermining cell growth does not give a measure of the process ofmetastasis. In fact, although uncontrolled growth is an important aspectof the initial events in the development of a cancer, the rate of growthof distant metastases can be remarkably slow. Hence the process ofmetastasis is largely independent of processes involving cell-growth,except in its final phases. Therefore, it is unlikely that oncogenes andtumour suppressor oncogenes will have much involvement in the process ofmetastasis and be useful diagnostic or therapeutic targets for controland elimination of metastatic disease.

It is one object of the present invention to identify DNA comprising,consisting of or containing sequences involved in metastasis,hereinafter referred to as metastasis inducing DNA's or Met-DNA's forshort.

According to a first aspect of the present invention there is provided amethod of screening and recovering a regulatory DNA capable of inducingmetastasis comprising the steps of:

-   -   i. Transferring tagged fragments of a human DNA from malignant,        metastatic cancer cells into a cell line that produces only        benign, non-metastasizing tumours when injected into a syngeneic        animal;    -   ii. injecting the transformed cells into the syngeneic animal;    -   iii. selecting those animals in which metastasizing tumours have        been identified; and    -   iv. recovering the regulatory DNA capable of inducing metastasis        therefrom.

Preferably the DNA fragments transferred in step 1 are fragments of from0.1 to 50 kilo base-pairs, more preferably 0.5 to 50 kilo base-pairs.

Preferably the cell line that produces only benign non-metastasizingtumours when injected into a syngeneic animal is a rat mammaryepithelial cell line, such as, for example Rama 37.

Preferably the fragments of human DNA from malignant, metastatic cancercells are tagged to assist in their removal or insertion from or into ahost or vector, such as, for example, the oligonucleotide tagillustrated in FIG. 1. This tagging procedure overcomes the problem ofidentifying the inserted human DNA sequences in the rat genome of thetransfected rat cells. Human-specific repetitive DNA (Alu) sequences arespaced sufficiently in the human genome that in many human DNA fragmentsof this size they will be absent.

In one embodiment, fragments of human DNA from malignant, metastaticbreast cancer cells are introduced into a rat mammary epithelial cellline Rama 37 which produces only benign, nonmetastasizing tumours wheninjected into syngeneic rats.

By way of example only, the transfer of restriction-enzymeHindIII-fragmented DNA from malignant metastatic rat and human breastcancer cell lines into a benign Rama 37 cell line produced a smallproportion (1-3%) of transformants which, when reintroduced into thesyngeneic rats, caused these cells to metastasise, principally to thelocal lymph nodes and lungs. In contrast, fragmented DNA fromnonmetastatic cells and the standard oncogenes (Ha-ras, Middle T Antigengene, and Large T Antigen gene) produced no metastasizing transformants.The latter result confirms the non involvement of such oncogenes in themetastatic process per se. However, the fact that metastasis can betransferred in a genetically dominant manner suggests that otherdominantly-acting DNA fragments are largely responsible for thisprocess. The full results of the above experiments are shown in table 1,which shows the incidence of tumours and metastases for Rama 37transfected cell lines.

The column headed “cells injected” gives the cell type in short hand,and full details are given below:

Rama 37 are Rat mammary 37 benign cells; R37-Ca2-LT1 is a cell line froma lung metastasis of Rama 37 cells transfected with fragmented DNA fromthe metastatic breast carcinoma cell line Ca2-83 (Cancer Res 542785-2795, 1994); B-T1 is a cell line from a primary tumour of Rama 37cells transfected with fragmented DNA from the benign breast cell lineHMT-3522 (Cancer Res. 54 2785-2795, 1994); R37-Ca2-HT is a cell line ofRama 37 cells transfected with tagged DNA fragments from metastatictransformant R37-Ca2-LT1; R37-Ca2-H is a cell line of Rama 37 cellstransfected with untagged DNA fragments from metastatic transformantR37-Ca2-LT1; R37-B-HT is a cell line of Rama 37 cells transfected withtagged DNA fragments from the benign transformant B-T1 as a control;R37-F1 is a cell line of Rama 37 transfected with PCR fragment F1 from acell line of a lung metastasis of R37-Ca2-HT; and R37-F2 is a cell lineof Rama 37 transfected with PCR fragment F2 from the same cell line of alung metastasis of R37-Ca2-HT.

The b annotation in the column headed metastases identifies thetransfecting DNA's giving rise to significantly more metastasis thanRama 37 cells (P<0.05, Fisher exact test). The animals were autopsiedafter 3 months.

To aid the rescue of metastasis-inducing human DNA sequences from therat transformant cell lines, all the HindIII-fragmented DNA's from onesuch metastatic transformant, R37-Ca2-LT1 (Table 1) were tagged at bothends with double-stranded synthetic oligonucleotides that providerestriction enzyme and unique PCR primer sites. These are shown in FIG.1 The tagged DNA fragments include 4 restriction sites: SfiI and NotI, adefective HindIII site at the 3′ end for linking to the HindIII sites atthe ends of the human DNA fragments, thereby destroying it, and aninternal HindIII site located near to the 5′ end, which when cut afterligation generated new fragments with HindIII ends. The fragments weretransfected into the parental Rama 37 cells, and after transfer of thecells to the mammary glands of syngeneic rats, metastatic cell lineswere isolated from the resultant rat lung metastases. The tagged,fragmented DNA incorporated into the metastatic transfected Rama 37 celllines was directly amplified between the tags by PCR and yielded bandsat about 1300 to 1500 bp that were responsible for the metastasizingability of the transfected cells. These results are shown in FIG. 2which shows the DNA fragments produced by PCR of metastatictransformants. Two new cell lines, established from the culture of lungmetastases of R37-Ca2-HT (tagged, metastatic DNA transformant) andR37-Ca2-H (untagged, metastatic DNA transformant) (see Table 1) in ratswere termed HTLu and HLu, respectively. They were run against the taggedbenign transformant cell line R37-B-HT and the tagged metastatictransformant R37-Ca2HT. Cellular DNA was amplified by PCR using a shortoligonucleotide primer of 22 bp from positions 3-24 of the tag sequenceas shown in FIG. 1. Compared with the control DNA's from HLu and B-HTcells, two extra bands, F1 and F2, of about 1300 bp and 1500 bprespectively, were specifically amplified from genomic DNA of the Ca2-HTand HTLu cells when PCRed DNA samples were run on 0.8 % agarose gelscontaining ethidium bromide and photographed in U.V. light. Thefluorescent bands of DNA are shown in negative imaging for clarity.Cloning of these pooled DNA's yielded six independent fragments and theresults are illustrated in FIG. 3. FIG. 3 shows pBluescript clones ofmetastatic DNA fragments F1 plus F2. The two broad PCR DNA fragments F1and F2 were excised from the gel in FIG. 2, combined, and cloneddirectly using the AT procedure into a suitably modified pBluescriptvector and the clones of recombinant vectors were cut with HindIII toexcise the cloned fragments. These cut recombinant vectors were analysedon a 0.8% agarose gel containing ethidium bromide and photographed inU.V. light. The sequences of some clones eg. C10 and C9-DNA's wereidentical; the six independent sequences arose from clones numberedC2,C5,C6,C9,C12 and C20 and hence are referred to as C2-DNA, C5-DNA etcas shown in FIG. 3. The position of the vector (Vec) DNA and insert(Ins) DNA are indicated and a standard molecular weight ladder—inkilobase pairs (kbp) is shown in lane M. Transfection of these clonedDNA fragments singly into the parental benign cell line confirmed thatall fragments (C2,C5,C6,C9,C12 and C20-DNA's) produce metastases. Theseare shown in Table 2 which tabulates the incidence of tumours andmetastases for Rama 37 cells transfected with cloned Met-DNA's. Thesuperscript a-e indicate:

^(a)Benign nonmetastatic Rama 37 cells were transfected with pSVneo orwith pSV2neo and different independently-cloned inserts of thepBluescript library of pooled F1- and F2-DNAs termed C2-DNA etc. or witha cylomegalovirus expression vector pBKCMV (CMV-1) or with the CDNA forosteopontin (opn) cloned into the same expression vector pBKCMVopn(OPN-1).

^(b)Transfectants were tested for their level of opn mRNA relative tothat in Rama 37 cells by Northern hybridisations to opn CDNA using aShimadzu CS9000 scanning densitomer. RNA loading levels werestandardised with respect to a 36B4 ribosomal protein constitutiveprobe.

^(c)Transfectants were tested in the mammary glands of rats for thepercentage (%) of tumour-bearing animals with metastases in the lungsafter 3 months. The incidence of tumours produced by all transfectantswas 100%.

^(d)Significantly higher levels than for Rama 37 cells (P<0.05; MannWhitney U test).

^(e)Significantly more metastases than for Rama 37 cells (P<0.05; Fisherexact test).

Thus Koch's postulate has been satisfied for allmetastasis-inducing-DNA's (Met-DNA's) in this system.

Southern hybridisations, and PCR amplifications have established thatthe Met-DNA's are specifically present in their respectivetransformants.

FIG. 4 shows detection of C9-DNA in transformant cell lines. CellularDNA was isolated from (A) a cell line from a lung metastasis producedinjection of C9-DNA transfected Rama 37 cells in rats; (B) C9-DNAtransfected Rama 37 cells (see FIG. 3 and Table 2); (C) benign Rama 37cells; (D) benign BT-1 cells (see Table 1). These DNA's were digestedwith HindIII and the digested DNA was analysed on 0.8 % agarose gelseither by (A) Southern blotting to a probe of [³²p] radioactivelylabelled C9-DNA, and the radioactivity visualised on X-ray film or (B)by PCR using the oligonucleotide fragment from either end of the C9-DNAas primers and run with a standard molecular weight marker ladder. Thenewly synthesised DNA in B is visualised by fluorescence of the ethidiumbromide in the gel in U.V. light.

Surprisingly, the sequences of these Met-DNA's (sequence 1 to 6hereafter), although human in origin, do not correspond to known genesand most do not include any known open reading frames. Furthermore noneof these Met-DNA's are expressed as mRNAs in their transformants andhence are not dominantly-acting oncogenes. They therefore containentirely novel short stretches of regulatory DNA capable of inducingmetastasis.

The state of the Met-DNA's has been investigated in the metastasizingtransformant cells. Bands of greater than 23 kbp which hybridise to theC9-DNA probe have been obtained from HindIII digested C9-DNAtransformants, and pulsed-field gel electrophoresis yields multiplebands of about 16-48 kbp after similar digestions as shown in FIG. 5a-d.

FIG. 5 shows the detection of Met-DNA in transformant cells. Thecellular DNA was isolated from: (A) cell line from a lung metastasisproduced by injection into rats of C9-DNA transfected Rama 37 cells; (B)C9-DNA transfected Rama 37 cells; (C) benign Rama cells; (D) benignprimary tumours of R37-BT-1 cells. These DNAs were digested with excessHindIII and the digested DNA was analysed on agarose gel (a) withcontinuous electric field; (b) with a pulsed electric field; or (c) byPCR using 17 mer oligonucleotide primers from each end of the C9-DNA;(d) These DNAs were also digested with excess EcoRl and analysed onagarose gels with a continuous electric field. The resultant gels wereeither (a.b.d) Southern blotted to a probe of [³²p] C9-DNA without tagsand the radioactivity visualised on X-ray film or (c) the newlysynthesized DNA was visualised by fluorescence of the bound ethidiumbromide in U.V. light. Controls with (a) C9 DNA in lane P and (c)standard molecular weight marker ladder in kilobase paris (kbp) in laneM were also run. This result strongly suggests that the flanking HindIIIsites have been destroyed by the transfection/integration process. Thehighest 48 kbp band is preferentially retained by the cell line isolatedfrom a lung metastasis (FIG. 5 b); thus is is likely that thisrepresents most of the metastasis-inducing DNA (Table 2). The C9-DNAtransfectants contain about 100 copies per haploid genome of C9-DNA whencompared with a single copy (FIG. 5 a, lane P) 10 copy and a 100 copyDNA control. PCR amplification of the integrated DNA using primerscomplementary to the cDNA adjacent to the untagged ends of C9-DNAproduces a single 1 kbp product showing that the integrity between theprimer sites has been maintained (FIG. 5 c). However, digestion of theDNA of C9-DNA transfectants with EcoRl (which cuts once internallywithin the C9-DNA) and hybridisation with a C9-DNA specific probe yieldspredominantly a 1 kbp band of similar size to the original C9-DNA insert(FIG. 5 d). This 1 kbp band probably arises from the digestion of tandemrepeats of C9-DNA. Similar results have been obtained with C2, C5, C6,C12 and C20-DNAs.

The occurrence of C9-DNA has been investigated in pilot studies in theDNA of human breast cancers. Hybridisation of C9-DNA occurs toHindIII-digested DNA from 4 out of the 9 breast tumours tested, whereasno hybridisation signal is detected from similarly-digested DNA fromnormal human breast or colon tissue. In this case a single hybridisingband of 1000 bp is detected (FIG. 6).

FIG. 6 illustrates detection of C9-DNA in human breast tumours. CellularDNA was isolated from a selection of nine randomly-picked human breasttumours numbered 14-130 and from normal breast and colon tissue togetherwith C9-DNA as a control. These DNAs were digested with an excess ofHindIII and the digested DNA was analysed on agarose gels, Southernblotted on to a filter and hybridised to a probe of [³²P]C9-DNA withouttags and the radioactivity visualised on X-ray film. Similar resultshave been obtained using PCR for C9-DNA.

According to a second aspect of the present invention there is provideda regulatory DNA capable of inducing metastasis consisting essentiallyof a human DNA fragment of less than 1.6 kilobase pair in lengthobtained from a malignant, metastasis cancer cell.

According to a third aspect of the present invention there are providedDNA consisting essentially of a regulatory DNA capable of inducingmetastasis from sequence 1: CTTCCTTGGT GCTCTATGTC TTGCCTCTCC CCTTCTCCAGTCCCATTAAG CCATAACCAT CTTGACAGAC TCTGGGACAG TCCCCTCTGC TCTCCTGTTGGCGCCTGAGT CCCTTTTTGC CTGAGGACCC TTCACGTAGC CTCCCATCTG GATGACCTAGTAGAAGACGT GGGAAGTTGT CACACTCAGG TAACTGAGCA GAGCTCAGAG ATTTAAAGTGAGTCTGGGGA GCCTCGAGGA TTGATCTGCT GCCTTAAAAA GCCAATTGGA TGACTAACCCAGACTATTGT CACTTTAGGT GGGAAGTCAC TAGCATATCT GATGGGTCAC ATCTGAGAAAGGTTTCTAGC AGTGGTGGCC TTGTGTGAGC AGCATGGCGT GTATCATGGT GTGCAGCATACTCAGGCTGC TTGCAACACT CGAGGCTCTT CTTCAGTATT AGGGGAACCA CTGGTGTTSGAACATGGTCC AGAATACAG  TCATGTGAGG AGAATCCCAA TGCGTCAGGA GAAAACGAGAGTCTGTGACC TCCATTCTTC AAGATACAGA ATTATTCTTG GACTGTGTTT TCATGCTCCTTGTGGATGGG AGTGAGTTTA CTTCAGGTTA ATGAGCATTG CTTACTGTTG GTATTCAAGTAAATGCTTAA ATTATCCTGG ATATACCTCT GTGGGAAGCA GGTTTTTGAT ACATGCAGCTTGTCCTTGTG ATTGATACTG CTTGAACTCA AGAGAACTTT GCTCATGTGA TCTTTCTTAACCGATGGAGT AGAAACTGTC TGATGCTCTC AATAAAGTTG GCTCTTGCAC GAGACGTTAGTCTGTCCTGT TTATCTGCTC CATTCTTCCG CTCCCACGGC CTCTACAGCA CTAAACCCACCACCGATAGA CTCAGTCTTT CACTGACAAA CATCACCAGA GGCTCTTAAC TGAGATTATAAACTGTTACT AGATGATGGG TTGAATCGCT CCCCAGAAAC ATAAACATTT ACTTGGAGAACTCAAGACCC CTTTGTAGAC ATAACTCCCA TGGT

According to a fourth aspect of the present invention there are providedDNA consisting essentially of a regulatory DNA capable of inducingmetastasis from sequence 2: ATTGCTGTGA GCCTATTAGC GACATTTGGT GACGCCCCTTTTAAGGGGGT AGATACAAAG AATGGGTTGA AATTCTGTGC CACAAACGCT CTCCATGTTTTCACAATTAC ACTTGCAACC TGTGGTCAGC AGCCAGAATT TAGGGATGTG ATGGGACAGGGTCGGGGAAA GAAGGAGAAG GGTAAAGGAA AGACAGCACG TTAAAGTCCA AACAGCTCCAGGAGACTATC TGTAGAAATA ACATCAGACC ATGAGGAGAA TTGATATCAT TGTTTTTCAATGGGTATCGC CAAGGGAACT TTCCATCTGA TTAAAAATAA TTACTGCTGG CACTAAATCCAATTGGAAAT GCCCCACACA ATTTATCTTC CACTTCATGC TGCTACCATA TGCCTGACGTGGCGGAGCAG AAGCATTCCC TCCCGTTCTG ATAAATAGTA CTTTGTAAAT ATTTGGAGACGGGAGCTCTG GTGACAGGGA ACACGTACAA ACCGGCCTGT TTATCATGTT CCCGATAGAGGCCCTCTTTG ACGTACAGGA CCCCAAAACA GTCAGGATGC TGTGAATTTC CTTCCATGAAGCCTTGTTCA CAATTAGCAA CCATTGGAGG AAGCAGGCTG CACTGTCTAC CACAAGTGGCACTTTCCAAA GAGGACACAT ATATTGGAGC AAGACATTTT GCTGGCTGAC TGGTGCTGTGTAAGCTGATA AACTGCTATA TTTATTAAAC TGGCTTTTCT TTGAACACCC CACTCAAGGAAAAAAAAACA CACTTAGGGT GACATTATTT GGAGATGAAG TCTTTATAGA GATGCTTAAGTTTAAACGAG ACTTTTAAAG CCGGCTCTAT TCCATTTAAT GAATGGTGTC CCTACAAAGGAAGAAACTGG GACAGAGGTA TGTACACTTG TGTGTGTGTG AGAGACAACG TGAGGAGCTGAAGAGGAGCA CGTACAAGTC AGAGAAAGGC TGACCCTTAT TCACACTGAG CAAACCAGTCATGTGTGGGT CGATAGATGA GAGTATCCCC CAAGACTCAC ACATTCGAAC GCTTGGTC

According to a fifth aspect of the present invention there are providedDNA consisting essentially of a regulatory DNA capable of inducingmetastasis from sequence 3: AGGACCAGAG TTCACATCCC ATCAAATGGC CCAGAAGGTTTTAATGCTGT CTTTTGGCCC AGGGGCGAAC TGCACACACA TGTGCACATA CACTTACAGAGACACACATT CAGCAGCATA AGAACACAAT CACAAATAAA AAAAATCTTG AAAAATTTTAAGCTAAAATT GTTAAGAAAT AACATATATA CAATTTTTCT TTATTTTTTT AAAGATTTATTTATTTAATG TATATGAGTA CACTGCCTCT CCCTCCAGAC ATAGCAGTAC AGGGCATCGGATCCCATTAC AGATGGTTGT GAGCCACCAT GTGGTTTCAC AGATGGTTGT GAGCCACCATGTGGTTTCAG GAATTGAACT CAGGACCTTT GGAAGAGCAG TCAGTGCTCT TAACCTCTAAGCCATCTCTC CTGACCCTTA TATACAATTT TAATGCTACG TACACACAAC TTCTCTTTCCTTTAATGGTT GAGATTTTTG TCTGGAGAAG TAAGAATAAA GGAGGGAAAG AACATTGCTTTCACATTGCA CCAGTGGGAA CAGCGTGTTT AAAGTAGGAA TGCCATGAAA TGACTGGCCTGCCTTCTCAT TACTGTTCCT CCCACTCCTC CTTTTAACTG GAGCTCCTTT ATCTAATTTATTAGTTTGAC GATACCCAGG GTTTTCTTCT GTTTTGATCT TTTTAAGACA GAGACTCACCATATAGCCCT GGCTGGCCTG AAGCTCACTA TGTAGACCAG TCTGGCCTTG AACTCAAAGGAGATCTATCT GCTTCCTAGT GCTGGGATTA AAGGCTTGTG CTACCAAGTC TGGTCTGAGGCTTTGGAGCA GCCTCGGTTT TGGCCTTCTT TAAGGATCTC TAAGCTAGCA GTAAGTAGCCTAGCCATGCT GTTGTAGGAA GTTGTTCGTT CATCCTGGCT CCAGCACAAA GGCAGTCACTAAACGTCGGC CTCATTTCAT CAGAGCTGAA TGCAAATTCC TTGTGCTCTT CCTGTGTCCTCCTGGAAC

According to a sixth aspect of the present invention there are providedDNA consisting essentially of a regulatory DNA capable of inducingmetastasis from sequence 4: AGTTGGGGAC ACAGCTTGCT TGATTAAGAT GTTTCTTGGGAAAAGGAGTT AAGCCTAATG ATTTCCAATG GAAAGGACTG CTAATTGGGG AGGCAATGTTGCTTAATTGG GACACCTGCG GGTAATTAAA AGCTCTCTCC CAGTGGCCTT TCCTGTTTTTGGCTCTGGGA GGCGAAGGCA TTGAGAGGGA TGCAGGCATT CTAAGGGCTG GTTCTTGGTTTCTCCCTTCC CCTCTGTCCA AACTCAGTGA GGTATCCCTG TCTGTGCTGT CCTTAGAGTGCCGTCCTGAG GCCTTGGTGA GTTAAGGTCT CTGGATCTGA GCTGCCTCAG GGAAACGCATGAGCTCATTG GAAAGGGGAG AACCAGGCAA AGGTGTTGGC TGTGACCTCA GAATTCTGAGGGGCAAAGGT TCAAGGCTAA CTCTCATTAT AGAGCAAGTT TGAGACTGGC CTGGGAACAAAAATATAAAG TGAGTGAGGT CATATGACAG CACCTGAGGA GTCCTGTCCC TAGAGATCATAAGGACCTGG CTGCTGGGGA CTTGTTGCAG ATGGCACTTT GTGTCGAGAG AGGGGACCTGCCCCAGCATG GGAGGCCCTG GAAGATCCTC TGGATTAACT GTGAACACTG ATTGCTGCTTTATACCTGGA GTTGTGCTGT TATCTGGTAC ACATCTGCTG GGTGAATGAG TTCATGGGCTTTATTTCAGT GAGGTATTTA CCTGAGGAGA AAGAAGGACT GGTGCCACAA AGCACAGCTTTTAAATCTGT GGGTTGTGAC CCATTATGGA CTATCATAAC TGAGTGCAGG TATCAAGAATACTTTAGCAG GTGGTAAAAA GATTTTTGAA TGCGCAACGA CCAAAACTGA ACTCAAAAATCAAGCATGGC ATGGATCCTG GGTGCTCCTG GAAGCACTTG CCTTTACTGC ATTGTGCGACTTGACGGTAG CCTTGGTTCT GAATGCACAA CACGTGGGCT TTGGGCTGCA CAGGCCACCACGCCGTGCCT GAAACACCTC AGCTCAGGTT TGTGGCTATG TCCTATGACT TGGACTTACTTTTATTGCAC ATATAAATAT TTTCCTGC

According to a seventh aspect of the present invention there areprovided DNA consisting essentially of a regulatory DNA capable ofinducing metastasis from sequence 5: GAGGGGGTGG TGGCACAGTT ATGTTTTTGTAGGAAGGGTT CCATGAACCT CAGCAGAGCT CGGGTTAGAA ATTTAAAAGC CCTGAGGGGAATTTTTTTTT TAAATCGCTA TGAATCTGAC ATGAGAAAAA CAGATCAGAA ACGTTCTTGTGCTTCAGAAA AGGACAAGTG TGTGAGCTAA CAGACTGCAC ACTGGTGTTC GAGGCACATCTGGATCACAG GAGCGTCAGA TAATGTCCCC AAAGGTAAAT GCATTTGCTT GCACAGTACCGAGTGTGGTG GGGGGTGCCT ACAGCCCAGC GGTTCTCAAC CTTCCTGATG CTTCGACCCTTTAATACAGT GCCTCATGCT CTGGTGACCT CCCCAACCTT AAAATTATTT TTGTTGCTGTTCATAACTGT GATTTTGATA CTGTTATGAA TTGTAATATA AATAATTTTG AAGAAAGAGGTTTGCCAAGG GTTTGAGAAC TGCTGTTCTA GCCCCACGTG GATGGTTTTT CGTCATTTGGGGTTTTTATG AGGCAGAGTC TTATGTAGCC CAGGCTAGCA GCCTAGAATG TGCTACTTAGCTGAGGAATA ACCTTGGAAC TTCTGAGGAC TGGAGAGACT GGCTTAGTCC TCAAGAAACTGGAAATAGCT GGAGTTTGGC TACTTGTGGG TTCCTTTTTC TTCAAACCTT TTCTACTCTTTTTCCACCCT GTCGGCCCCC TAACACTAAA TAAGAAAGAG AAAGGGGAGC ATAGAGGGGAAAAGAAACCC CTGAATAACG TCAGTAGTTG GCAAAGGGGG GTGACATATG TTGTCATTAGACCACATCCT GGTGATTAAG GGGAGTCAAG TTCCTTGGGG CAAGTTTGAT CTTTCGTGTAACGATATCTA ATTTCTTCTC CCTGTTGCTT CGTCTTTGTG AACAACGACT TGATAACCCACAATGGACCA TCAACCAACC AACCAACCAT

According to a eighth aspect of the present invention there are providedDNA consisting essentially of a regulatory DNA capable of inducingmetastasis from sequence 6: TTGTCTCTGG TGTTACTTGT TTTCCCATTT CTGACAGTGGTTTGACCTTC TATACGCCTG TGTGTCAGGA GTGCTGTAGA CCTATTTTCC TGTTTTCTTTCAGCCAGTTA CAGGAACAGA GTGTTCTACT GTCAGATGTG TAGCTGTTCC TGTCCACTGACTTTCAAGCT GTCTCTGTGT GCAGGAACCA GAAGGGCCTG TCCCTACTTC TACTGGGCCCCTACGCACAG GGGGCCTAGA TGGTGCTAGG TGTTTTCCTC TAGAGCCTGA AATGTGGGCAGAGAGTAGTC TCCTCTGGTT TCCTAGGTAT GTCTTCCCCT CTGAAGGTCT AGCTCTCCCTTCCATGGGAT ATGGGTGCAG GGAGCTGTTT GACCAGGTCC TCTCAAATCC GGGTGCAGTCTGGACCGCAG GCTCCTGTAG CTTGCCTGCT GCAATCTTCC CGCACCCAGA GGCACCCAAGTTTCCTCTTG GGCCAAGGAT GTGGGCAAAG GTGGGCAGAA GTGGCAATCT CTCCTGCCCTAGCGTCTCAG GATTGCCCTC ACTTCTGGGC AATCCGCTCT CTCTTCCACA GGGTTTGGGAGCAGGGAGCT GTGGGCCGGT ATCAGGCAAA GGTTTGAGGC AACCAGTTAG AAACTGGAAGTGTCAGGTCC CAGAGGAATT TTGCCTTTGT GTGTCCTGAG TCCACCAGGC AGGTCACTTGGAGCAGAAAA ATTGGTTTTC CCCTCGGTCT CAGGCCTGAA GTTGCACCTC AGGGTTGGCTTTCAGCTGTA CCTGTGGAAA GTATGGTTTT AAAAATCTAA GATAGCTATC ATGCAGCAAGGCTTGTGTAA AATGTCTATT TGGTTCCTTT ATGACTTACT TTTGCTGTAC TGAGGATCAAACCTAGGGTC TCAAGCAGTC ATCACAATTC TCTGTCACTG ATCCAGCTCC ATTTCTATTTTCTTTTGTCC CGCGCGATCT CTCGCCAGCA AGAAAACACG CTAGGGACAT ACGAATCCTTGCTGCAGCCA AAACTTTTAT TGAATCTTAA GGAGAAGCCC GCGCACCGGA CTGGCGCGGTTTATATACAC CCTAGCACAG TGCATCCACA

Detailed examination of there DNA sequences has confirmed that the sixMet-DNA's bear little relationship to one another. C6-DNA shows 86%homology to 102 bp of the rat WAP promoter (Nucleic Acids Res. 128685-8697 1984) with a novel duplication of 30 nucleotides of thisregion. All Met-DNAs contain recognition sequences for transcriptionfactors TCF-1 (EMBO J. 10. 123-132, 1991) and HIP1b (Mol.cell. Biol. 10,653-661, 1990). Moreover all but one contain recognition sequences forCTCF (Oncogene 5, 1743-1753, 1990), HIP1a (Mol.Cell.Biol.10, 653-661,1990), NF-1L6 (EMBO J. 9 457-465, 1990) and regions of potential Z-DNA(Nature 282, 680-686, 1979), with C6-DNA containing a tract of 23alternating purine-pyrimidine bases. Thus these novel sequences allcontain potential regulatory regions for transcription of DNA into mRNAbut no known coding or viral-related sequences.

According to an ninth aspect of the present invention there is providedthe use of an osteopontin gene as a metastasis inducing transformant.

In one embodiment Met-DNA's, are introduced into a benign rat mammaryepithelial cell line Rama 37.

By way of example and to help identify the regulatory function thatshort stretches of human malignant DNA (precursor to Met-DNA's) mayexert on the transfected Rama 37 cells, the mRNA expression of themetastatic transformant rat mammary cell line R37-Ca2-LT1 was comparedwith its benign parental cell line Rama 37 using subtractivehybridisation techniques. Of the four subtracted clones threecorresponded to known rat genes for proteins including osteopontin andone corresponded to a novel rat gene of unknown function. As an exampleonly, transfection of rat osteopontin cDNA into the parental Rama 37cells produced transformants that induced a high frequency of metastasiscompared with vector controls confirming the metastatic capability ofthe osteopontin gene as shown in Table 2.

These overall results have established a causal relationship between theMet-DNA's and metastasis on the one hand and the over-or underexpressionof certain rat genes, at least one of which is novel, that are linked tothe metastatic process in this rat system. Controls with DNA's fromnonmalignant, nonmetastatic sources as well as the oncogenes Ha-ras-l,Polyoma Large T Antigen and Polyoma Middle T Antigen failed to inducemetastasis establishing the specificity of the inductive processes inthis system.

At present the most useful indication of whether a breast or othercommon cancer will metastasise in the future in a patient is whether theprimary tumour has already spread to the local lymph nodes. This testonly works on a population basis. For example, in breast cancer, thereare many examples of patients with no tumour in the lymph nodes atpresentation who later die of metastatic disease and of patients withmetastatic deposits in the lymph nodes who live a normal life-span. Thusan accurate test of good predictive value for the occurrence ofmetastases would be important in selecting those patients for vigorousconventional chemotherapeutic treatments without causing the potentiallyharmful side-effects in those patients who do not need this treatment.

According to a tenth aspect of the present invention there is provided aprobe specific to regulatory DNA capable of inducing metastasis.

By specific is meant hybridises to any target DNA under suitable saltand temperature conditions to allow detection of identical or relatedDNA molecules.

Preferably the probe is provided as part of a kit which may additionallycomprise one or more of the following: a colour indicator; anoligonucleotide primer; materials for gel analysis, and/or materials forDNA transfer or hydridisation.

The Met-DNA sequences may be detected in tumour or biopsy specimens bystandard Southern blotting, PCR-based or in-situ techniques to identifythose patients at risk from metastatic disease. Physical methods ofdetection based on imaging techniques may also be possible. Expressionof metastasis-inducing genes may be detected by standard mRNAhydridisation PCR amplification or by antibodies specific for thegene-product.

According to a eleventh aspect of the present invention there isprovided a medicament adapted to target a regulatory DNA capable ofinducing metastasis as claimed in any of claims 7 to 13.

In one embodiment such Met-DNA's, metastasis-inducing genes or fragmentsthereof, could be targeted in the cancer cells to excise or block theirfunction using synthetic oligonucleotides based on a knowledge of thesequence of the Met-DNA's, metastasis-inducing genes or fragmentsthereof, of the invention.

In another embodiment such Met-DNA's, metastasis-inducing genes orfragments thereof, may be targeted for treatment using standard antibodyand antisense mRNA/ribozyme techniques for detection and fordestruction, respectively.

1. A probe specific to a regulatory DNA which is not expressed as anmRNA but is capable of inducing metastasis, said regulatory DNAconsisting essentially of a human DNA fragment of less than 1.5 kb inlength and comprising the sequence of SEQ. ID. NO. 4, obtained from amalignant, metastasis cancer cell, wherein said probe is less than 6000base pairs in length.
 2. A probe specific to a regulatory DNA which isnot expressed as an mRNA but is capable of inducing metastasis, whereinsaid regulatory DNA has the sequence of SEQ. ID. NO. 4 and wherein saidprobe is less than 6000 base pairs in length.